Method for preparing antitumor drug by inducing neuroblastoma differentiation through human embryonic stem cell and neuroblastoma cell co-culture supernatant

Abstract

The invention provides a method for preparing an antitumor drug by inducing differentiation of neuroblastoma through co-culture supernate of human embryonic stem cells and neuroblastoma cells. The human embryonic stem cells have the biological characteristics of multidirectional differentiation potential, infinite proliferation, self-renewal and the like, and the microenvironment of the human embryonic stem cells can reprogram cancer cells. The early neuroblastoma can naturally fade, and research shows that cancer cells of the neuroblastoma retain certain differentiation potential and can be differentiated towards neurons. The inventor discovers that the neuroblastoma cells and embryonic stem cells are subjected to direct contact co-culture, and the generated co-culture supernatant can induce the neuroblastoma cells to differentiate into neuronal cells, so that the proliferation, migration and invasion of the neuroblastoma cells are inhibited in vitro. The discovery shows that the co-culture supernate has huge potential in innovative biological treatment application of resisting neuroblastoma.

Description

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a method for preparing an anti-tumor drug by inducing the differentiation of neuroblastoma with the co-culture supernatant of human embryonic stem cells and neuroblastoma cells. Background Art

[0002] Neuroblastoma (NB) is an extracranial solid tumor caused by defects in differentiation due to genomic and epigenetic damage. It is one of the most common malignant tumors in children and is known as the "king of childhood tumors". Clinical symptoms vary depending on the location of the primary tumor, and common manifestations include abdominal mass, abdominal pain, respiratory distress, or neurological symptoms caused by spinal cord involvement. Although in some early cases of neuroblastoma, the lesion will regress spontaneously, high-risk patients tend to be highly invasive. Approximately 60% of high-risk children will relapse, and 20% will progress to refractory diseases, with a 5-year overall survival rate (OS) of only about 20%.

[0003] Neuroblastoma is a cancer developed from undifferentiated sympathetic ganglion cells. Therefore, differentiation induction therapy has great potential in the treatment of this tumor. Currently, retinoic acid (RA) is the most effective drug for inducing the differentiation of neuroblastoma, which can induce neuroblastoma cells to differentiate into a neuronal state, thereby reversing their malignancy. Studies have shown that neuroblastoma cell lines treated with RA not only have a significantly reduced proliferation ability but also an increased formation of neurites, indicating significant neuronal-like differentiation. However, due to the amplification of the MYCN gene in most high-risk neuroblastoma patients, RA treatment is prone to drug resistance, resulting in limited efficacy. Therefore, developing a biotherapy strategy based on differentiation induction to promote the transformation of neuroblastoma from malignant to benign not only has important clinical significance for the treatment of neuroblastoma but also has the potential to be extended to the treatment of other solid tumors after clarifying its molecular mechanism, providing a new direction for tumor treatment. Summary of the Invention

[0004] To solve the problems in the prior art, the present invention provides a method for preparing an anti-tumor drug by inducing the differentiation of neuroblastoma with the co-culture supernatant of human embryonic stem cells and neuroblastoma cells.

[0005] The inventors found that when neuroblastoma cells are added to the microenvironment of human embryonic stem cells, the human embryonic stem cells will interact with neuroblastoma, thereby secreting "differentiation-promoting factors", and then applying the extracted supernatant to the treatment of neuroblastoma. It was found that during the co-culture process, if the ratio of the two types of cells is inappropriate, the addition time point of neuroblastoma cells is inaccurate, or the collection time and concentration of the co-culture supernatant are not suitable, the human embryonic stem cells cannot exert the biological effect of promoting the differentiation of neuroblastoma and reversing tumor malignancy.

[0006] Unless otherwise specified, the parts mentioned in the present invention are by weight, and the percentages are by mass percentage.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] A method for inducing the differentiation of neuroblastoma into neurons by using the co-culture supernatant of human embryonic stem cells and neuroblastoma cells, which is characterized by comprising the following steps:

[0009] (1) Pre-coat the substrate gel for human pluripotent stem cells to prepare a special culture plate for human embryonic stem cells;

[0010] (2) Use the special culture plate for human embryonic stem cells pre-coated in step (1) to resuscitate and inoculate human embryonic stem cells;

[0011] (3) Passage human embryonic stem cells;

[0012] (4) Stimulate embryonic stem cells with neuroblastoma cells: Take human neuroblastoma cells in the logarithmic growth phase, wash the cells 2 times with PBS, then digest them with 0.25% Trypsin-EDTA (37 °C, 1 min 30 s), terminate the digestion with an equal volume of DMEM complete medium, centrifuge (5 min, 1000 rpm), discard the supernatant, add DMEM basal medium to resuspend into single cells, and add them to human embryonic stem cells with 50% confluence at a ratio of neuroblastoma cells: human embryonic stem cells of 1:1.5.

[0013] (5) Put the co-culture system of the two kinds of cells into a CO2 cell culture incubator, and collect the co-culture supernatant for 48 - 96 h.

[0014] The human embryonic stem cells described in the present invention are an established stem cell line.

[0015] The special medium for human embryonic stem cells is PGM1 human pluripotent stem cell medium (Beijing Saibei Biology CA1007500), Y-27632 (MCE Company, USA HY10071).

[0016] The Matrigel substrate gel is produced by Corning Company (354277). The preparation method of the special culture plate for human embryonic stem cells is as follows: After thawing the Matrigel substrate gel on ice, dilute it with DPBS buffer solution according to the ratio (Matrigel: DPBS / PBS buffer solution = 1:80), then spread it into the culture plate, and place it in a 37 °C, 5% CO2 culture incubator for 3 - 4 h until the substrate gel solidifies and forms, and then it can be used.

[0017] The method for resuscitating human embryonic stem cells is to rinse a special culture plate pre-coated with matrigel matrix gel once with DPBS, add 1 mL / well of human pluripotent stem cell complete medium, and pre-warm it in an incubator at 37°C with 5% CO2; take out human embryonic stem cells from liquid nitrogen, melt them in a 37°C constant temperature water bath, aspirate the cells into a 15 mL centrifuge tube pre-added with 2 mL of human pluripotent stem cell complete medium, centrifuge (5 min, 1000 rpm), after centrifugation, discard the waste liquid, add 1 mL of human pluripotent stem cell complete medium to resuspend into small cell clumps, evenly seed them in the pre-warmed culture plate coated with matrix gel, and change the medium every 24 h.

[0018] The method for subculturing the human embryonic stem cells is to rinse a special culture plate pre-coated with matrigel matrix gel once with DPBS, add 1 mL / well of human pluripotent stem cell complete medium, and pre-warm it in an incubator at 37°C with 5% CO2; aspirate the waste liquid from human embryonic stem cells in the logarithmic growth phase, add DPBS to wash once, then add 800 uL of human pluripotent stem cell digestive solution, place it in an incubator at 37°C with 5% CO2 for digestion for 4 min, after the digestion time, observe the cells under the microscope, the originally dense cell colonies become loose, the edges of the cell colonies turn white, and some cells begin to fall off, add 1 mL of human pluripotent stem cell complete medium to terminate the digestion, and transfer it to a centrifuge tube for centrifugation (5 min, 1000 rpm), discard the waste liquid, add 1 mL of human pluripotent stem cell complete medium to resuspend into small cell clumps, and evenly seed them in the pre-warmed culture plate coated with matrigel matrix gel.

[0019] The state of human embryonic stem cells matures when the cell confluence of embryonic stem cells reaches 50%-60%, the clone edges are smooth and there are no differentiated cells, the cells are dispersed among each other, and no fusion has occurred.

[0020] Collect the co-culture medium. After co-culturing for 48 h - 96 h, collect the co-culture supernatant with a centrifuge tube, centrifuge it, and then filter it with a microporous filter membrane to collect the supernatant.

[0021] Beneficial effects

[0022] Human embryonic stem cells (hESCs) are pluripotent stem cells obtained from the inner cell mass of blastocysts, with the potential to differentiate into any cell type in the human body. Their unique microenvironment can reprogram cancer cells, thus avoiding the side effects brought by traditional anti-cancer therapies. The inventor found that when neuroblastoma cells are added to the embryonic stem cell microenvironment, significant interactions will occur between the two, prompting embryonic stem cells to secrete "differentiation-promoting factors". Extracting this supernatant and applying it to tumor treatment can effectively induce the differentiation of neuroblastoma cells.

[0023] The "differentiation-promoting factor" produced by human neuroblastoma-stimulated embryonic stem cells in the present invention can significantly induce the differentiation of neuroblastoma cells into neurons and reduce their stemness, thereby inhibiting the proliferation, migration, and invasion of tumors, indicating that the co-culture supernatant has great potential in innovative biotherapy against neuroblastoma. Brief Description of the Drawings

[0024] Figure 1 It is a figure showing the results of an experiment on the induction of the differentiation of neuroblastoma cells into neurons by the co-culture supernatant, specifically a cell morphology experiment result figure;

[0025] Figure 2 It is a figure showing the results of an experiment on the induction of the differentiation of neuroblastoma cells into neurons by the co-culture supernatant, specifically an experiment result figure for detecting the expression of the neuronal marker β-III tubulin by immunofluorescence staining;

[0026] Figure 3 It is a figure showing the results of an experiment on the induction of the differentiation of neuroblastoma cells into neurons by the co-culture supernatant, specifically an experiment result figure for detecting the mRNA expression of tumor stemness markers and neural differentiation markers by qRT-PCR;

[0027] Figure 4 It is a figure showing the results of the in vitro proliferation inhibition of neuroblastoma cells by the co-culture supernatant;

[0028] Figure 5 It is the inhibitory effect of the co-culture supernatant on the migration ability of neuroblastoma cells, specifically a figure showing the results of a cell Transwell migration experiment;

[0029] Figure 6 It is the inhibitory effect of the co-culture supernatant on the migration ability of neuroblastoma cells, specifically a statistical chart of the results of a cell Transwell migration experiment;

[0030] Figure 7 It is the inhibitory effect of the co-culture supernatant on the invasion ability of neuroblastoma cells, specifically a figure showing the results of a cell Transwell invasion experiment;

[0031] Figure 8 It is the inhibitory effect of the co-culture supernatant on the invasion ability of neuroblastoma cells, specifically a statistical chart of the results of a cell Transwell invasion experiment. Detailed Embodiments

[0032] The present invention will be specifically described below through specific embodiments. It should be noted here that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above-mentioned invention content. The raw materials and reagents used in the present invention are all commercially available products. Among them, the neuroblastoma in the embodiment is the neuroblastoma SH-SY5Y cell line, which is preserved by the Laboratory of Biochemistry and Molecular Pharmacology, Chongqing Medical University; the human embryonic stem cells in the embodiment are the established stem cell lines, specifically, the human embryonic stem cell H9 cell line is purchased from the Chinese Academy of Sciences Stem Cell Bank.

[0033] Example 1

[0034] Preparation of co-culture supernatant of human embryonic stem cells and neuroblastoma cells

[0035] Prepare a special culture plate for human embryonic stem cells by pre-coating Matrigel matrix gel: After thawing the Matrigel matrix gel on ice, dilute it with DPBS buffer solution according to the ratio (Matrigel: DPBS / PBS buffer solution = 1:80) and spread it into the culture plate. Place it in an incubator at 37°C and 5% CO2 for 3-4 hours until the matrix gel solidifies and forms, then it can be used.

[0036] Digestion and passage: Take out the complete medium for human pluripotent stem cells and equilibrate it to room temperature. Take out the culture dish coated with Matrigel matrix gel, aspirate the bottom working solution and add an appropriate amount of complete medium for human pluripotent stem cells, and place it in a 37°C constant temperature CO2 cell incubator. Select human embryonic stem cells in good condition (that is, hESCs with a cell coverage rate of about 80%, large stem cell colonies, regular clone edges and no differentiated cells). After aspirating the medium, add 1 mL of DPBS to wash for 20-30 s, then add 800 μL of human pluripotent stem cell digestive solution, and place it in a 37°C constant temperature and 5% CO2 incubator for digestion for 4 minutes. After the digestion time is up, observe the cells under the microscope. The originally dense cell colonies become loose, the edges of the cell colonies turn white, and some cells begin to fall off. Add 1 mL of complete medium for human pluripotent stem cells to terminate the digestion, and transfer it to a centrifuge tube for centrifugation (5 minutes, 1000 rpm). Discard the waste liquid, add 1 mL of complete medium for human pluripotent stem cells to resuspend it into small cell clumps, and evenly plant it in the pre-warmed culture plate coated with Matrigel matrix gel.

[0037] Co-culture: Human neuroblastoma cells in the logarithmic growth phase were taken. After washing the cells twice with PBS, they were digested with 0.25% Trypsin-EDTA (37 °C, 1 min 30 s), and the digestion was terminated with an equal volume of complete DMEM medium. After centrifugation (5 min, 1000 rpm), the supernatant was discarded and the cells were resuspended in DMEM basal medium to form single cells. The neuroblastoma cells and human embryonic stem cells were added to the human embryonic stem cells with 50% confluence at a ratio of 1:1.5.

[0038] Preparation of co-culture supernatant: The co-culture supernatant was collected in a 15 mL centrifuge tube for 48 - 96 h, centrifuged, and then filtered through a 0.22 μm microporous filter membrane. The filtered supernatant was aliquoted and stored in 5 mL sterile Eppendorf tubes at -80 °C.

[0039] Example 2

[0040] The co-culture supernatant of human embryonic stem cells and neuroblastoma cells can promote the differentiation of neuroblastoma and inhibit its stemness

[0041] 1. Cell morphology

[0042] Neuroblastoma cells in the logarithmic growth phase were taken and evenly seeded in six-well plates at a density of 5×10 5 cells per well. After 24 h, all the cells adhered to the wall, and the medium was replaced with the corresponding co-culture supernatant and hESC supernatant, etc., and cultured for 72 h. The results showed ( Figure 1 ), that there was obvious neurite growth in the experimental group compared with the other control groups.

[0043] 2. Immunofluorescence staining

[0044] Neuroblastoma cells in the logarithmic growth phase were taken and evenly seeded in twelve-well plates with coverslips at a density of 2×10 5 cells per well. After adhesion, the medium was replaced with the corresponding co-culture supernatant and hESC supernatant, etc., and cultured for 72 h. The coverslips with growing cells were washed twice with PBS and fixed with 4% paraformaldehyde (Sigma) at room temperature for 30 minutes. After washing twice with PBS, the cells were incubated with mouse anti-β-III tubulin antibody overnight at 4 °C. The next day, after washing the cells twice with PBS, they were incubated with TRITC-conjugated goat anti-mouse IgG antibody for 2 hours at room temperature in the dark. Finally, the cells were stained with DAPI (VectorLabs) and mounted on slides for observation under a fluorescence microscope. The results showed ( Figure 2 ), that compared with the Control group, RA and hESC-CM groups, the cells treated with the co-culture supernatant had obvious neurites expressing β-III tubulin, indicating that it significantly induced the differentiation of neuroblastoma.

[0045] 3. qRT-PCR

[0046] Use a 6-cm 2 culture dish to culture neuroblastoma cells. When the cells reach 60% density, replace the culture medium with co-culture supernatant and hESC supernatant and continuously act for 72 h. Extract cellular template RNA, reverse transcribe it into cDNA, and perform qRT-PCR experiments: After designing the primer sequences, dilute the upstream and downstream primers with DEPC water, prepare the premixed system Mix, sequentially add DEPC water, primers, and syber green, add the mixed solution Mix to the eight-well strip tube on an ice box, add cDNA to each well in turn, and detect the amplified products by amplification curve analysis method. GAPDH is used as an internal reference, and all data are analyzed by the 2 -△△Ct method. The results show ( Figure 3 ), after neuroblastoma cells are stimulated with co-culture supernatant for 72 h, the mRNA content levels of its differentiation-related indicators are significantly increased, and the tumor stemness-related indicators are down-regulated.

[0047] 4. Cell proliferation experiment

[0048] Take neuroblastoma cells in the logarithmic growth phase and inoculate 5×10 3 cells per well into a 96-well plate. Add 100 μL of co-culture supernatant with volume fractions of 0%, 20%, 40%, 60%, and 80% respectively. Place the 96-well plate in an incubator at 37 °C. After 24 h, add 10 μL of CCK8 working solution to each well for about 2 h, and measure the absorbance value of each well at 450 nm with an enzyme-linked immunosorbent assay reader. Cell survival rate = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%. The results show ( Figure 4 ), the co-culture supernatant can inhibit the proliferation of neuroblastoma cells and its inhibitory effect is concentration-dependent.

[0049] 5. Cell migration experiment

[0050] Take neuroblastoma cells in the logarithmic growth phase that have starved for 24 h, digest, centrifuge, and resuspend the cells with the co-culture supernatant of embryonic stem cells stimulated by neuroblastoma cells and the supernatant of embryonic stem cells. Inoculate 5×10 4 cells per well into the upper chamber of a Transwell chamber, and use complete medium containing 20% FBS in the lower chamber. After 48 h, aspirate the liquid in the upper chamber, fix it with 4% paraformaldehyde for 20 min, then stain it with crystal violet solution for 20 min, wash off the excess crystal violet solution with PBS, and observe and count the number of cells passing through the chamber membrane under the microscope. The results show ( Figures 5-6 ) compared with the Control group and the hESC-CM group, the co-culture supernatant has a stronger ability to inhibit migration in vitro.

[0051] 6. Cell invasion experiment

[0052] Pre-coat 100 μL of Matrigel in the upper chamber and place it in an incubator at 37 °C with 5% CO2 for 4 h. Take neuroblastoma cells in the logarithmic growth phase that have been starved for 24 h, digest, centrifuge, and resuspend the cells with the co-culture supernatant of embryonic stem cells stimulated with neuroblastoma cells and the supernatant of embryonic stem cells. Seed 5×10 4 cells into the upper chamber of a Transwell chamber, and use complete medium containing 20% FBS in the lower chamber. After 36 h, aspirate the liquid in the upper chamber, fix with 4% paraformaldehyde for 20 min, then stain with crystal violet solution for 20 min, wash away the excess crystal violet solution with PBS, and observe and count the number of cells that have passed through the chamber membrane under the microscope. The results show that ( Figures 7-8 ) compared with the Control group and the hESC-CM group, the co-culture supernatant has a stronger ability to inhibit invasion in vitro.

Claims

1. A method for preparing an anti-tumor drug by inducing neuroblastoma differentiation using the supernatant of co-culture of human embryonic stem cells and neuroblastoma cells, characterized in that: The steps include: (1) Pre-laying human pluripotent stem cells on a base of matrix gel to prepare a special culture plate for human embryonic stem cells; (2) using the human embryonic stem cell culture plate pre-coated in step (1) to perform human embryonic stem cell recovery and inoculation; (3) Human embryonic stem cells are passaged; (4) Stimulation of embryonic stem cells with neuroblastoma cells: Human neuroblastoma cells in the logarithmic growth phase were taken, washed twice with PBS, digested with 0.25% Trypsin-EDTA (37°C, 1 min 30 s), and digested with an equal volume of DMEM complete medium. The cells were centrifuged (5 min, 1000 rpm), the supernatant was discarded, DMEM basal medium was added to resuspend the cells into single cells, and the cells were added to 50% confluent human embryonic stem cells at a ratio of 1:1.5 for neuroblastoma cells and human embryonic stem cells. (5) Place the two cell co-culture systems in a CO2 cell culture incubator and collect the co-culture supernatant for 48-96 hours.

2. The method according to claim 1, characterized in that: The human embryonic stem cell-specific culture medium is PGM1 human pluripotent stem cell culture medium (Beijing Saibei Biotechnology CA1007500) and Y-27632 (American MCE Company HY10071).

3. The method according to claim 1, characterized in that: The Matrigel matrix glue is produced by Corning (354277). The preparation method of the special culture plate for human embryonic stem cells is as follows: after thawing the matrigel matrix glue on ice, dilute it with DPBS buffer in proportion (matrigel: DPBS / PBS buffer = 1:80) and spread it on the culture plate, and place it in a 37°C, 5% CO2 incubator for 3-4 hours until the matrix glue solidifies and takes shape before use.

4. The method according to claim 1, characterized in that: The human embryonic stem cell recovery method is to rinse the special culture plate coated with matrigel matrix gel in advance with DPBS, add 1mL / well human pluripotent stem cell complete culture medium and put it into a 37°C constant temperature, 5% CO2 incubator for preheating; take out human embryonic stem cells from liquid nitrogen, melt them in a 37°C constant temperature water bath, and absorb the cells into a 15mL centrifuge tube to which 2mL human pluripotent stem cell complete culture medium has been added in advance, centrifuge (5min, 1000rpm), after centrifugation, discard the waste liquid, add 1mL human pluripotent stem cell complete culture medium to resuspend into small cell clumps, and evenly plant them in the preheated culture plate coated with matrix gel, and change the liquid every 24h.

5. The method according to claim 1, characterized in that: The human embryonic stem cell passage method comprises the following steps: washing a special culture plate pre-coated with matrigel matrix gel with DPBS, adding 1 mL / well of human pluripotent stem cell complete culture medium and placing the plate in a 37°C constant temperature, 5% CO2 incubator for pre-warming; taking human embryonic stem cells growing in the logarithmic phase, removing waste liquid, adding DPBS for washing, adding 800uL of human pluripotent stem cell digestion solution, placing the plate in a 37°C constant temperature, 5% CO2 incubator for digestion for 4 minutes, and after the digestion time is up, observing the cells under a microscope, and finding that the originally dense cell colonies become loose, the edges of the cell colonies turn white, and some cells begin to fall off, adding 1 mL of human pluripotent stem cell complete culture medium to terminate the digestion, transferring the plate to a centrifuge tube for centrifugation (5 minutes, 1000 rpm), discarding the waste liquid, adding 1 mL of human pluripotent stem cell complete culture medium to resuspend the plate into small cell clumps, and planting the cells evenly in the pre-warmed culture plate coated with matrigel matrix gel.

6. The method according to claim 5, characterized in that: The mature state of human embryonic stem cells is when the confluence of embryonic stem cells reaches 50%-60%, the clone edge is smooth and there are no differentiated cells, the cells are scattered, and no fusion occurs.

7. The method according to claim 1, characterized in that: The co-culture solution is collected after 48h-96h of co-culture, and the co-culture supernatant is collected using a centrifuge tube, centrifuged, and then filtered using a microporous filter membrane to collect the supernatant.

8. Use of the co-culture solution prepared by the method according to any one of claims 1 to 7 in the preparation of anti-neuroblastoma drugs.

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